Pivotal roles of interleukin-6 in transmural inflammation in murine T cell transfer colitis -- Kitamura et al.

76 (6): 1111 -- Journal of Leukocyte Biology

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Originally published online as doi:10.1189/jlb.0604328 on August 31, 2004

Published online before print August 31, 2004

(Journal of Leukocyte Biology. 2004;76:1111-1117.) This Article

© 2004 by Society for Leukocyte Biology
Abstract
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Kazuya Kitamura*, ,1, Yasunari Nakamoto*, Shuichi Kaneko* and Naofumi
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ABSTRACT
TOP
Breakdown of normal mucosal immunity is one of the major causes for
ABSTRACT
inflammatory bowel disease. Interleukin (IL)-6 is a proinflammatory cytokine INTRODUCTION
produced aberrantly in various types of inflammation, but its role in inflammatory MATERIALS AND METHODS
bowel disease is still obscure. Hence, we analyzed the roles of IL-6 in the RESULTS
pathogenesis of murine T cell transfer colitis, whose histopathology resembles DISCUSSION
Crohn’s disease. The transfer of CD4+CD45RBhigh T cells into severe combined REFERENCES
immunodeficiency mice induced the infiltration of T cells and macrophages, and
the gene expression of CC chemokine receptor (CCR)1, CCR2, CCR5, CXC chemokine receptor 3, their ligands, tumor
necrosis factor- , interferon- , and IL-6 was progressively augmented as colitis developed. The incidence of transmural
colitis was significantly reduced with a minimal decrease in the severity of colitis in recipients transferred with CD4

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 Pivotal roles of interleukin-6 in transmural inflammation in murine T cell transfer colitis -- Kitamura et al. 76 (6): 1111 -- Journal of Leukocyte Biology

+CD45RBhigh T cells derived from IL-6-deficient mice compared with those with wild-type mice. Moreover, the gene
expression of several cytokines, chemokines, and matrix metalloproteinases was reduced significantly in recipients
transferred with IL-6-deficient, mice-derived T cells. These observations suggested that T cell-derived IL-6 may augment
the gene expression of several proinflammatory molecules, thereby causing transmural inflammation. Thus, IL-6 might be
a promising target for treating transmural inflammation in Crohn’s disease, which can lead to severe complications such
as strictures, fissures, and fistulas.

Key Words: inflammatory bowel disease • animal model • chemokine • matrix metalloproteinase

INTRODUCTION
TOP
Breakdown of normal mucosal immunity is presumed to contribute to the
ABSTRACT
development of inflammatory bowel disease (IBD), encompassing two major INTRODUCTION
1 2
forms, Crohn’s disease and ulcerative colitis [ , ]. Crohn’s disease is MATERIALS AND METHODS
characterized by transmural inflammation, resulting in stricture, fissure, or fistula RESULTS
1 DISCUSSION
in the gastrointestinal tracts [ ]. It was reported that several proinflammatory
cytokines such as tumor necrosis factor (TNF)- and interleukin (IL)-6 were REFERENCES
aberrantly produced, resulting in enhanced levels in sera or colon of the patient
345
with Crohn’s disease [ ]. Accumulating evidence has revealed that treatment with Infliximab, a chimeric monoclonal
678
antibody (mAb) against TNF- , has been effective in the patients with refractory Crohn’s disease [ ]. Moreover, Ito
9
and his colleagues [ ] reported that treatment with anti-IL-6 receptor (IL-6R) mAb has also been effective in the patients
with Crohn’s disease. However, a precise role of these molecules has not been clarified yet.

3 10 11
A number of animal models have been proposed to clarify the pathogenesis of IBD [ , , ]. When severe combined
immunodeficient (SCID) mice received CD4+CD45RBhigh T cells, they developed transmural colitis resembling human
12 13 14
Crohn’s disease [ ]. Hence, this model was frequently used to clarify the pathogenesis of Crohn’s disease at
molecular and cellular levels. Accumulating evidence indicates that transferred T cells play roles in several aspects,
15 16
including the impairment of mucosal immunity in T cell transfer colitis [ , ]. Even in this model, various
proinflammatory molecules, such as TNF- and IL-6, were presumed to be aberrantly produced. However, the precise
roles of IL-6 in this animal model have been obscure yet.

Recently, two studies revealed that anti-IL-6R antibody ameliorated colitis in a mouse model of Crohn’s disease.
17
Yamamoto and his colleagues [ ] suggested that the effect of anti-IL-6R antibody was associated with decreased
18
expression of proinflammatory cytokines or adhesion molecules. Conversely, Atreya and his colleagues [ ] speculated
that anti-IL-6R antibody decreased the expression of an antiapoptotic gene, including Bcl-2 and Bcl-xL, resulting in
resistance of apoptosis in inflammatory cells and perpetuation of inflammation. Moreover, another group demonstrated
that IL-6 contributed to establishment of murine colitis through an activating signal transducer and activator of
19
transcription-3/Janus kinase pathway [ ]. However, the cellular origin of IL-6 in this model has not been proven.
Moreover, it remains to be investigated about the effects of donor-derived IL-6 on the infiltration of inflammatory cells
and chemokines regulating the infiltration steps.

Here, we determined the chemokine and cytokine gene expression as well as the cell infiltration during the course of CD4

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 Pivotal roles of interleukin-6 in transmural inflammation in murine T cell transfer colitis -- Kitamura et al. 76 (6): 1111 -- Journal of Leukocyte Biology

+CD45RBhigh T cell transfer colitis. Moreover, we examined the roles of T cell-derived IL-6 in this model by
reconstituting SCID mice with IL-6-deficient, mice-derived T cells. We provided the evidence to suggest that T cell-
derived IL-6 can crucially be involved in the pathogenesis of transmural inflammation in this colitis model.

MATERIALS AND METHODS

TOP
Mice
ABSTRACT
Specific pathogen-free BALB/c and C.B-17 SCID mice were purchased from
INTRODUCTION
Charles River Japan Co. (Yokohama) and maintained in the Animal Facility of
MATERIALS AND METHODS
Kanazawa University (Japan). BALB/c mice were designated as wild-type (WT) RESULTS
mice in the following experiments. IL-6-knockout (KO) mice were generated as DISCUSSION
20
described previously [ ] and back-crossed to BALB/c mice for more than eight REFERENCES
generations. All mice were female and used at the age of 6–8 weeks. All animal
experiments were performed in accordance with the Guideline for the Care and Use of Laboratory Animals on the Takara-
machi Campus of Kanazawa University.

Antibodies
For a flow cytometric or immunohistochemical analysis, the following antibodies were used. Purified and phycoerythrin
(PE)-conjugated rat anti-mouse CD4 (L3T4, clone RM4-5) and fluorescein isothiocyanate (FITC)-conjugated rat anti-
mouse CD45RB (clone 16A) mAb were purchased from BD PharMingen (San Diego, CA). Rat anti-mouse F4/80 (clone
A3-1) and rat anti-mouse DEC205 (clone NLDC-145) mAb were obtained from Serotec (Oxford, UK). Rabbit anti-
myeloperoxidase (MPO) polyclonal antibodies were obtained from NeoMarkers (Fremont, CA). Rat anti-mouse IL-6
21
mAb (clone 6B4) was prepared as described previously [ ]. Isotype-matched immunoglobulin G (IgG) from each animal
was used as a negative control.

Induction of T cell transfer colitis

12 13
T cell transfer colitis was induced as described previously with minor modifications [ , ]. Briefly, CD4+ T cells were
enriched from splenocytes obtained from BALB/c mice by using anti-CD4 (L3T4) MACS® magnetic beads (Miltenyi
Biotec, Auburn, CA) according to the manufacturer’s instructions. Thereafter, CD4+CD45RBhigh cells were sorted on a
Coulter EPICS® Elite (Beckman Coulter, Fullerton, CA) after a double-color immunostaining with anti-CD4 and anti-
CD45RB antibodies. The CD45RBhigh population was defined as the most brightly stained cells consisting of 40–50% of
CD4+ cells. The purity of sorted cells was 96–99% on the reanalysis. Five hundred thousand cells in 200 µl phosphate-
buffered saline were injected intravenously into each recipient SCID mice. All procedures were conducted under aseptic
conditions.

Histological and immunohistochemical examinations

The colon was harvested from recipient mice before or 1, 3, 5, and 7 weeks after T cell reconstitution. For a histological
examination, 10% neutral-buffered, formaldehyde-fixed, and paraffin-embedded sections were deparaffinized and stained
with hematoxylin and eosin (H&E). The degree of inflammation on a microscopic cross-section of the colon was graded
by an examiner without any knowledge about the experimental procedures, semiquantitatively from 0–4, as described
22
previously [ ] with minor modifications: 0, no signs of inflammation; 1, low level; 2, moderate level of leukocyte
infiltration; 3, high level of leukocyte infiltration, loss of goblet cells, thickening of the colon wall; and 4, high level of
leukocyte infiltration with ulceration or transmural inflammation. Six coronary sections were assessed, and the sum of
each histological score was shown as an individual score, 0–24.

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For an immunohistochemical identification of the types of infiltrating cells, frozen sections were prepared and fixed with
cold acetone for 2 min. After being treated with 1% (v/v) hydrogen peroxide in methanol, the sections were incubated
overnight at 4°C with the primary antibody. The tissue sections were then rinsed and further incubated with biotin-
conjugated, anti-rat IgG antibody and treated with Vectastain Elite ABC kit (Vector, Burlingame, CA) according to the
manufacturer’s instructions. The slides were then reacted with Vectastain DAB substrate kit (Vector) and counterstained
with hematoxylin.

For a double-color immunofluorescence analysis of CD4 and IL-6, frozen sections were prepared and fixed with 10%
neutral-buffered formaldehyde for 15 min. The sections were incubated for 2 h at 37°C with anti-IL-6 antibody. The tissue
sections were washed and incubated with FITC-conjugated anti-rat IgG antibody. Then, the sections were washed and
further incubated with PE-conjugated anti-CD4 antibody and examined under a fluorescence microscope.

RNA isolation and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

TM
Total RNA was extracted from a part of colon tissues with RNA-Bee (Tel-Test Inc., Friendswood, TX), according to
23
the manufacturer’s instructions, and cDNA was synthesized as described previously [ ]. Briefly, 2 µg total RNA was
reverse-transcribed at 30°C for 10 min, at 42°C for 60 min, and at 99°C for 5 min in 20 µl reaction mixture containing 100
units ReverTra Ace® (Toyobo, Osaka, Japan) and 50 pmol hexanucleotide random primers (Takara Shuzo Co., Shiga,
Japan). Thereafter, 0.5 µl cDNA was amplified in 25 µl PCR reaction mixture containing 5.0 nmol deoxy-unspecified
nucleoside 5'-triphosphate mixture (Takara Shuzo), 8.25 pmol primers, 2.5 µl tenfold PCR buffer (Takara Shuzo), and
0.625 units recombinant Taq DNA polymerase (Takara Shuzo). All primers used were purchased from Genset Oligos
(Paris, France). The amplification was performed using a GeneAmp® PCR System 9700 (Perkin-Elmer, Foster City, CA)
23
under the conditions as described previously [ ]. Under these conditions, the quantity of amplified products did not
reach a saturated level. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium
bromide under an ultraviolet illumination on a GelDoc 2000 (Bio-Rad, Hercules, CA). The intensities of the bands were
measured with the aid of National Institutes of Health Image Analysis software (Version 1.62, National Institutes of
Health, Bethesda, MD), and the ratio to ß-actin was determined. The relative intensities were calculated on the assumption
that the ratio of SCID mice, reconstituted with WT mice-derived T cells 5 weeks ago, was 1.00.

Statistical analysis
Statistical significance of colitis scores was analyzed by the Mann-Whitney’s U-test. The incidence of transmural
inflammation was compared by the 2 test. The relative intensity of PCR products was analyzed using one-way ANOVA
followed by the Fisher’s protected least significant difference test. P values lower than 0.05 were considered statistically
significant.

RESULTS
TOP
Colitis development in SCID mice reconstituted with CD4+CD45RBhigh T
ABSTRACT
cells
INTRODUCTION
We first examined pathological changes in SCID mice reconstituted with CD4
MATERIALS AND METHODS
+CD45RBhigh T cells derived from WT mice in a time-kinetic manner. There were
RESULTS
no histopathological changes in the colon of recipient mice until 1 week after the DISCUSSION
reconstitution (Fig. 1a 1b and 1f ). Colonization of reconstituted T cells in the REFERENCES

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recipient’s lymphoid system was evidenced by T cell zone formation in the spleen of recipient mice 1 week after the
reconstitution (data not shown). At 3 weeks after the reconstitution, inflammatory cells infiltrated into the lamina propria
of the recipient’s colon, and mild crypt elongation was observed (Fig. 1c) . Inflammatory cell infiltration became more
evident 5 weeks after the reconstitution, and moderate-to-severe crypt elongation was observed with a frequent
development of transmural inflammation or ulceration (Fig. 1d and 1f) . At 7 weeks after the reconstitution, severe colitis
with transmural inflammation appeared in a substantial portion of mice reconstituted with WT mice-derived CD4
+CD45RBhigh T cells (Fig. 1e) .

Figure 1. Histological changes in C.B-17 SCID mice reconstituted with CD4

+CD45RBhigh T cells. Colon tissues were harvested from recipient mice
before (a) or 1 (b), 3 (c), 5 (d), and 7 (e) weeks after the reconstitution and
were stained with H&E. All animals were examined histologically, and
representative results from at least five animals at each time-point are shown
here. Original magnification, x150. The degree of inflammation in the colon
was assessed semiquantitatively from 0 to 4, as described in Materials and
Methods. Six coronary sections were assessed, and the sum of each
histological score was shown as an individual score, 0–24 (f). Each symbol
represents the histological score of one individual animal, and each bar
represents the median of each group. (•) Animals with transmural
inflammation.
View larger version (88K):
[in this window]
[in a new window]

CD4+ T cells and F4/80+ macrophages predominantly infiltrated into the inflamed colon
To determine the types of infiltrating cells, we performed an immunnohistochemical analysis. There were several CD4-
positive and F4/80-positive cells, a few DEC205-positive cells, and no MPO-positive cells in the colon until 1 week after
the reconstitution (Fig. 2b 2g 2l and 2q ). At this time-point, CD4- and DEC205-positive cells were mainly localized in
lymphoid aggregations (Fig. 2b and 2l) , and F4/80-positive cells were in lymphoid aggregations as well as lamina propria
(Fig. 2g ). At 3 weeks after the reconstitution, mild inflammatory cell infiltration was observed within lamina propria of
the colon, and the major population of infiltrating cells was CD4-positive T cells and F4/80-positive macrophages (Fig. 2c
and 2h) . The infiltration became more evident 5 and 7 weeks after the reconstitution (Fig. 2d 2e 2i and 2j) . Moreover,
DEC205-positive dendritic cells were distributed in lamina propria as well as lymphoid aggregations in the inflamed but
not noninflamed colon (Fig. 2k 2l 2m 2n 2o) . On the contrary, MPO-positive neutrophils were scattered in the inflamed
colon later than 5 weeks after the reconstitution (Fig. 2p 2q 2r 2s 2t) .

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Figure 2. Immunohistochemical detection of CD4 (a–e)-, F4/80 (f–j)-,

DEC205 (k–o)-, and MPO-positive cells (p–t) in the colon. Colon tissues
were obtained from recipient mice before (a, f, k, p) or 1 (b, g, l, q), 3 (c,
h, m, r), 5 (d, i, n, s), and 7 weeks (e, j, o, t) after the reconstitution and
were immunostained as described in Materials and Methods. At least
three animals were killed at each time-point for immunostaining, and
representative results are shown here. Original magnification, x200.

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[in this window]
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Gene expression of chemokines, their receptors, and cytokines in the colon of recipient mice
The presence of numerous infiltrating cells in the inflamed colon prompted us to analyze the gene expression of
chemokines and chemokine receptors by a semiquantitative RT-PCR, as chemokines are presumed to regulate leukocyte
migration. CC chemokine ligand (CCL)2, CCL3, CCL4, CCL5, CCL22, CXC chemokine ligand (CXCL)2/3, CXCL9,
CXCL10, and CXCL11 gene expression was significantly and progressively increased as colitis progressed (Fig. 3 ).
Concomitantly, the gene expression of the receptors for these ligands, CCR1, CCR2, CCR4, CCR5, CXCR2, and
CXCR3, was also enhanced (Fig. 3) . These observations suggested the potential involvement of these chemokines in the
inflammatory cell migration in this model.

Figure 3. Changes in chemokine, chemokine receptor, and cytokine gene

expression in the colon of C.B-17 SCID mice reconstituted with CD4
+CD45RBhigh T cells derived from WT mice. Total RNA was extracted
from the colon of recipient mice before or 1, 3, 5, and 7 weeks (w) after
the reconstitution, and RT-PCR was performed as described in Materials
and Methods. Three individual animals were examined in each group.
The ratio of each PCR product to ß-actin was determined, and the
relative intensity was calculated as described in Materials and Methods.
View larger version (32K): The relative intensities were compared between the untreated group and
[in this window] each group. Results are expressed as mean, and error bars indicate SEM.
[in a new window] *, P < 0.01; **, P < 0.05. CCR, CC chemokine receptor; CXCR, CXC
chemokine receptor; IFN- , interferon- ; TGF-ß, transforming growth
factor-ß.

As inflammatory cells produce various cytokines, we next analyzed the cytokine gene expression in the inflamed colon.
The gene expression of IL-6, TNF- , IFN- , and IL-12p40 was significantly increased, in line with the previous studies
14 24
(Fig. 3) [ , ]. Although the T helper cell type 1 (Th1)-immune response was presumed to be predominant in this
model, the gene expression of IL-18, one of the Th1 cytokines, was not enhanced during the course of the disease.

Detection of IL-6-producing cells in the inflamed colon

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4 5
As serum IL-6 levels were reported to be increased in patients with Crohn’s disease [ , ], and IL-6 mRNA expression
was progressively enhanced as the disease advanced in this model (Fig. 3) , we next analyzed IL-6-producing cells in the
colon of the recipient mice 5 weeks after the reconstitution. IL-6-positive cells were detected in the inflamed colon (Fig.
4b ), whereas there were few IL-6-expressing cells in the colon of control mice (Fig. 4e) . Most of the IL-6-positive cells
were CD4-positive (Fig. 4c) . Thus, we speculated that transferred T cell should be an essential producer of IL-6 in the
inflamed intestine in this model.

Figure 4. A double-color immunofluorescence analysis for CD4 and IL-6

expression. Colon tissues were harvested from recipient mice before (d,
e, f) or 5 weeks after (a, b, c) the reconstitution and immunostained for
CD4 (a, d) or IL-6 (b, e) as described in Materials and Methods. Merged
images are also shown (c, f). At least three animals were killed for the
immunofluorescence analysis, and representative results are shown here.
Original magnification, x200.

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[in this window]
[in a new window]

Transmural inflammation was attenuated in the colon of SCID mice reconstituted with IL-6-KO, mice-derived T
cells
To elucidate the pathophysiological roles of IL-6, we analyzed the histological changes in the colon of SCID mice
reconstituted with CD4+CD45RBhigh T cells from IL-6-KO mice. Mild-to-moderate colitis was observed 5 weeks after
the reconstitution from IL-6-KO donors (Fig. 5b ). The major population of infiltrating cells was also CD4-positive T
cells and F4/80-positive macrophages in the IL-6-KO group (data not shown). The median of colitis score was higher in
the WT group than the IL-6-KO group, albeit without any statistical significance (Fig. 5c ; P=0.38). However, five of 10
(50%) mice manifested transmural inflammation or ulceration in the WT group, whereas no transmural inflammation was
observed in the IL-6-KO group (Fig. 5c ; P<0.05). At 7 weeks after the reconstitution, no transmural inflammation was
also observed in the IL-6-KO group (data not shown). These findings indicated that T cell-derived IL-6 may essentially be
involved in the development of transmural inflammation.

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Figure 5. Histological changes in C.B-17 SCID mice reconstituted with

CD4+CD45RBhigh T cells from WT (a) or IL-6-KO (b) donors. Colon
tissues were harvested from recipient mice 5 weeks after the
reconstitution and were stained with H&E. Ten animals in each group
were examined histologically, and representative results are shown here.
Original magnification, x150 (a, b). The degree of inflammation in the
View larger version (42K): colon was assessed semiquantitatively from 0 to 4 as described in
[in this window] Materials and Methods. Six coronary sections were assessed, and the sum
[in a new window] of each histological score was shown as an individual score, 0–24 (c).
Each symbol represents the histological score of one individual animal,
and each bar represents the median of each group. (•) Animals with
transmural inflammation.

Gene expression of proinflammatory molecules in the colon of recipient mice reconstituted with IL-6-deficient
donor T cells
To determine why transmural inflammation was attenuated in the absence of T cell-derived IL-6, we compared gene
expression of several proinflammatory molecules between WT and IL-6-KO groups at 5 weeks after the reconstitution. As
shown in Figure 6 , mRNA levels of CCL2, CCL3, CCL4, CCL5, CCL22, CXCL2/3, CXCL11, CCR2, CCR4, CXCR2,
and CXCR3 were significantly decreased in the IL-6-KO group, compared with the WT group (Fig. 6) . Moreover, gene
expression of TNF- and IFN- was significantly attenuated in the IL-6-KO group. Furthermore, IL-6 gene expression
was markedly decreased in the inflamed colon of the KO group, compared with the WT group (Fig. 6) . These findings
indicated that transferred T cells can be a major source of IL-6 in the inflamed colon and that other types of cells,
including macrophages or stromal cells, also produced IL-6. Additionally, mRNA levels of MMP-3 and MMP-9 were
significantly reduced in the IL-6-KO group compared with the WT group (P<0.01). These observations suggested that T
cell-derived IL-6 may regulate the gene expression of chemokines, proinflammatory cytokines, and MMPs, thereby
inducing the development of transmural inflammation.

Figure 6. Gene expression of proinflammatory molecules in the colon of

C.B-17 SCID mice reconstituted with CD4+CD45RBhigh T cells from
WT or IL-6-KO mice. Total RNA was extracted from the colon of
recipient mice 5 weeks after the reconstitution. Three individual animals
from each group were examined. The ratio of each PCR product to ß-
actin was determined, and the relative intensity to WT mice was
calculated. The relative intensities of WT and IL-6-KO groups were
compared with untreated mice. Results are expressed as mean, and error
View larger version (37K): bars indicate SEM. *, P < 0.01; **, P < 0.05. MMP, Matrix
[in this window] metalloproteinase.
[in a new window]

DISCUSSION
We observed that the expression of several proinflammatory cytokines such as TNF- and IL-6 was significantly

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augmented in the inflammatory site of T cell transfer colitis, in line with the TOP
14 24 25
previous reports [ , , ]. Therapies targeted for TNF- or IL-6 significantly ABSTRACT
9 INTRODUCTION
alleviated inflammation in Crohn’s disease as well as animal models of colitis [ ,
17 18
, ]. These observations suggest that IL-6 as well as TNF- are good MATERIALS AND METHODS
RESULTS
molecular targets for preventing and/or treating IBD. However, it still remains
DISCUSSION
elusive about the cellular sources or the precise functions of IL-6 in the colitis.
REFERENCES
Hence, we undertook adoptive transfer of IL-6-KO mice-derived CD4
+CD45RBhigh T cells into SCID mice. The incidence of transmural colitis was
remarkably reduced with a marginal decrease in the severity of colitis in the IL-6-KO group compared with the WT
group. IL-6, which was produced by recipient-derived cells (Fig. 6) , may obscure the effects of the lack of donor-derived
IL-6. Nevertheless, these observations suggest that donor-derived IL-6 was indispensable for the induction of transmural
inflammation.

14 26 27
Several lines of evidence [ , , ] and our findings implicated Th1 cells as a crucial cell component in the induction
of T cell transfer colitis. The effects of IL-6 on Th cell polarization are controversial. IL-6 can suppress Th1 polarization
28
by up-regulating suppressor of cytokine signaling-1 and eventually interfering with IFN- signaling [ ]. On the contrary,
several lines of evidence indicated that IL-6 was crucially involved in Th1 response-mediated pathologies including
29 30
experimental autoimmune encephalomyelitis [ ] and Candida albicans infection [ ]. In line with the latter
observations, IFN- but not IL-4 gene expression was attenuated in the IL-6-KO group when compared with the WT
group. These observations suggest that IL-6 can promote Th1 polarization in this T cell transfer colitis model.

We observed that CD4+ T cells started to infiltrate 3 weeks after the reconstitution, before the appearance of apparent
12 13
pathological changes in the colon, consistent with the previous reports [ , ]. Concomitantly, the gene expression of
specific sets of chemokines, such as CXCL9, CXCL10, CXCL11, CCL3, CCL4, and CCL5, was enhanced later than 3
31 32
weeks after the reconstitution, consistent with the previous reports [ , ]. CXCL9, CXCL10, and CXCL11 can bind
with CXCR3, and CCL3, CCL4, and CCL5 can bind with CCR1 and CCR5. Moreover, the gene expression of CXCR3
33 34
and CCR5, which are presumed to be expressed predominantly on Th1 cells [ , ], was also enhanced in the inflamed
colon. We provided the first and definitive evidence about the synchronous occurrence of inflammatory cell infiltration
and chemokine/chemokine receptor expression in this animal model of Crohn’s disease. These observations may support
the notion that these chemokines should be involved in Th1 cell migration into the intestine and subsequent establishment
of colitis, based on the observation that the expression of CXCR3, CCR5, and their ligands was enhanced in colons from
35
patients with Crohn’s disease [ ].

F4/80-positive macrophages accumulated in the inflamed intestine along with T cells. Concomitantly, the inflamed
intestine exhibited enhanced gene expression of chemokines such as CCL2, CCL3, CCL4, and CCL5, which can recruit
macrophages as well as T cells. Moreover, enhanced gene expression of these chemokines was significantly reduced in
the IL-6-KO group compared with the WT group. The absence of IL-6 depressed CCL2 production and eventually
36
reduced T cell or macrophage infiltration [ ], although the precise molecular mechanism has not been determined.
37 38
CCL2, CCL3, and CCL4 gene transcription can be negatively repressed by the same transcription factor, BCL-6 [ , ].
Thus, it is tempting to speculate that IL-6 may regulate this transcription factor and eventually the gene expression of
these chemokines.

Our findings provided evidence to indicate the crucial roles of T cell-derived IL-6 in the pathogenesis of transmural
inflammation. A characteristic feature of Crohn’s disease, transmural inflammation, leads to severe complications such as

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39
strictures, fissures, and fistulas [ ], which sometimes need surgical treatment. A recent study revealed that anti-TNF-
40
antibody was effective to treat fistulas of Crohn’s disease patients [ ], although its precise molecular mechanism has not
been determined yet. MMPs are a family of zinc-containing endoproteinases, which degrade extracellular matrix and are
41 42
deeply concerned in tissue remodeling [ , ]. Enhanced activities of MMP-2, MMP-3, and MMP-9 were observed on
43 44 45
IBD of human [ ]. Moreover, increased MMP-3 gene expression was documented on the colon of patients with
46
Crohn’s disease but not with ulcerative colitis on cDNA microarray analysis [ ]. Furthermore, Tarlton and colleagues
47
[ ] observed the association of MMP-2 and -9 with transmural inflammation in T cell transfer colitis. We also observed
that MMP-3 and -9 gene expression was enhanced in the inflamed colon and that the enhanced expression was
significantly reduced in the IL-6-KO group. TNF- can induce IL-6 production in various types of cells, and IL-6 can
48 49
induce the expression of MMP in various types of cells [ , ]. Thus, it is likely that anti-TNF- antibodies may inhibit
the production of IL-6, which can regulate the expression of MMPs, crucial molecules in the development of transmural
inflammation.

Our present results demonstrated that T cell-derived IL-6 has a pivotal role in the pathogenesis of transfer colitis,
particularly transmural inflammation, by inducing the expression of proinflammatory cytokines, chemokines, and MMPs.
Thus, IL-6 may be a promising target for treating Crohn’s disease, especially with transmural inflammation, which can
lead to severe complications such as strictures, fissures, and fistulas.

ACKNOWLEDGEMENTS
This work is supported in part by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and
Technology. We thank Dr. Jacque Van Snick (Ludwig Institute for Cancer Research, Brussels, Belgium) for kindly
providing hybridomas for anti-IL-6 mAb (6B4). We express our gratitude to Dr. Toshikazu Kondo (Wakayama Medical
University, Japan) for his technical advice about a double-color immunofluorescence analysis.

Received June 1, 2004; accepted July 30, 2004.

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